A key characteristic of human and experimental heart failure is impaired calcium cycling through the sarcoplasmic reticulum (SR), which contributes to deteriorated contractility. The major functions of the SR are: Ca-uptake mediated by SERCA2a and its reversible regulator phospholamban (PLN); Ca-storage through calsequestrin and the histidine rich Ca-binding protein (HRC); and Ca-release occurring through the ryanodine receptor complex. Our central hypothesis is that alterations in the fine-tuned regulation of SR Ca-cycling play a critical role in heart failure progression. The long-term goal of this project is to determine the triggers and mechanisms, which disrupt SR function, to reveal new therapeutic targets. Our previous studies showed that phospholamban interacts with SERCA2 and inhibits its Ca-affinity, representing a fundamental brake in SR Ca-cycling and cardiac contractility. Thus, inhibition of PLN activity to normalize the depressed Ca- homeostasis has been suggested to be of therapeutic benefit in failing hearts. However, we have recently shown that phospholamban also interacts with the anti-apoptotic HS-1 associated protein X-1 (HAX-1). Actually, HAX-1 enhances the inhibitory effects of phospholamban on SERCA2a, while isoproterenol stimulation relieves this inhibition. In addition, we uncovered that HRC interacts with SERCA2a and regulates the maximal rates of Ca-transport. Thus, we have identified a multimeric complex (HAX- 1/PLN/SERCA2a/HRC), which mediates SR Ca-uptake in the heart. However, the functional significance of these newly identified regulators of SR Ca-cycling is not currently clear. We propose here to: a) define the role of HAX-1 by temporal regulation of its expression levels in the adult heart to assess its function on SR Ca- cycling and cardiomyocyte apoptosis under physiological and stress conditions; and b) elucidate the functional significance of HRC ablation and the human S96A-HRC variant in cardiac SR Ca-uptake and release as well as overall cardiomyocyte function. We will employ an integrated approach with studies at the molecular, biochemical and physiological levels. Our preliminary results in each of the specific aims make the proposed studies attractive and feasible, especially since changes in the levels or activity of Hax-1 and HRC are expected to be linked to changes in cardiac function and/or cell death. These studies will provide further fundamental insights into the role of SR Ca-handling in cardiac physiology and pathophysiology. PUBLIC HEALTH RELEVANCE A universal characteristic of the failing hearts is depressed calcium cycling through the sarcoplasmic reticulum, which reflects deteriorated heart function. This proposal concentrates on elucidating the role of two proteins involved in sarcoplasmic reticulum calcium cycling: HAX-1, which has been known to regulate cell survival; and the histidine rich calcium binding protein (HRC), which is present in the sarcoplasmic reticulum lumen. Our studies will clearly advance our knowledge on these two protein players involved in cardiac function and dysfunction, which may lead to better therapeutic avenues in heart failure.